Project Summary Half of human cancers overexpress a Long Interspersed Element-1 (LINE-1, L1) encoded protein, open reading frame 1 protein (ORF1p). Though typically silenced by CpG methylation in somatic cells, L1 promoters become hypomethylated in human cancers, ultimately leading to a `reawakening' that generates somatic L1 insertions into cancer genomes. This has been described in many types of gastrointestinal tract cancers, as well as lung and ovarian cancers. L1 retrotransposition (RTn) requires ORF1p and a second protein, ORF2p, which possesses endonuclease and reverse transcriptase activities. ORF2p endonuclease cleaves target site DNA and reverse transcriptase copies L1 RNA into the genome in a process known as target primed reverse transcription (TPRT). ORF2p endonuclease activity is associated with double-strand breaks (DSBs) in cultured cells, and L1 expression is cytotoxic in cell culture. This proposal seeks to reconcile the growth deficits induced by L1 overexpression in vitro with the prevalent expression of ORF1p in malignant cells in vivo. I hypothesize that expression of L1 results in ORF2p nucleolytic activity that causes DSBs in both cultured cells and in cancers. Further, ORF2p-generated DSBs induce cytotoxicity in non-transformed cells. Aim 1 proposes to map loci in the genome bound by ORF2p and provide evidence for ORF2p-mediated DSBs at these sites, establishing a direct link between L1 expression and DNA damage. Aim 2 seeks to elucidate the genetic determinants of cellular fitness in L1-expressing cells and is broken down into two parts. Aim 2A tests the hypothesis that p53 restricts RTn and survival in non-transformed cells expressing L1. Aim 2B will identify genes contributing to L1 toxicity by implementing an unbiased genome-wide knockout screen, and includes a rigorous validation strategy. Together, these studies will provide insights into the mechanisms that enable cancer cell survival and proliferation in the face of L1 expression.